Abstract:

The invention relates transiently attaching drag-tags to molecules during
electrophoresis. The invention includes running buffers having drag-tags
that transiently attach to lipophilic moieties attached to the molecules.
The lipophilic moieties can be covalently or ionically bonded to the
molecules. One particular aspect of the invention is a nucleoside analog
or a nucleic acid analog comprising a lipophilic moiety. The invention is
also directed to methods of separating molecules that comprise a
lipophilic moiety. The methods generally comprise transiently attaching a
drag-tag to the lipophilic moiety during a separation modality. These
methods can be used to separate the molecules by size or weight, to
measure a hydrodynamic radius of a drag-tag, or to separate a plurality
of drag-tag by their hydrodynamic radius.

Claims:

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. A nucleoside analog comprising a nucleoside bonded to a lipophilic
moiety comprising an alkyl group selected from the group consisting of an
octyl, a nonyl, a decyl, an undecyl, a dodecyl, a tridecyl, a tetradecyl,
a pentadecyl, a hexadecyl, a heptadecyl, an octadecyl, a nonadecyl, an
icosyl, a henicosyl, a docosyl, a tricosyl and a tetracosyl.

7. The nucleoside analog according to claim 6, further comprising a sugar
moiety having a 3' end, wherein the lipophilic moiety is bonded to the 3'
end of the sugar.

8. The nucleoside analog according to claim 6, further comprising a sugar
moiety having a 5' end, wherein the lipophilic moiety is bonded to the 5'
end of the sugar.

9. The nucleoside analog according to claim 6, wherein the lipophilic
moiety comprises a functional group.

10. The nucleoside analog according to claim 9, wherein the functional
group is a chromophore.

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. A method of transiently attaching a drag-tag to a molecule
comprising:a. providing a molecule comprising a polar moiety and a
lipophilic moiety; andb. moving the molecule through a running buffer
comprising a drag-tag, wherein the drag-tag comprises a structure, and
wherein the structure comprises a surfactant, a polymer or a combination
thereof.

16. The method according to claim 15, wherein the lipophilic moiety is an
alkyl group.

17. The method according to claim 16, wherein the alkyl group selected
from the group consisting of an octyl, a nonyl, a decyl, an undecyl, a
dodecyl, a tridecyl, a tetradecyl, a pentadecyl, a hexadecyl, a
heptadecyl, an octadecyl, a nonadecyl, an icosyl, a henicosyl, a docosyl,
a tricosyl and a tetracosyl.

18. The method according to claim 15, wherein each lipophilic moiety
comprises an approximately equal number of carbon atoms.

22. The method according to claim 15, wherein the structure is selected
from the group consisting of a liposome, a micelle, a solid particle, a
carbon nanotube, and an oil-in-water emulsion.

23. The method according to claim 15, wherein the molecule comprises a
nucleic acid analog.

24. The method according to claim 15, wherein the molecule comprises a
protein-detergent complex.

25. The method according to claim 15 further comprising:c. applying an
electric field to the running buffer for a period of time;d. forming a
lipophilic interaction between the lipophilic moiety and the drag-tag for
a portion of the period of time;e. terminating the lipophilic interaction
formed during step (d) during the period of time;f. repeating steps (d)
and (e) throughout at least a portion of the period of time; andg.
discontinuing the electric field.

26. (canceled)

27. (canceled)

28. (canceled)

29. The method according to claim 15, wherein the lipophilic moiety
comprise a functional group.

30. The method according to claim 29, wherein the functional group is a
chromophore.

31. The method according to claim 15, wherein lipophilic moiety is
selected from the group consisting of sodium dodecyl sulfate, sodium
lauryl sulfate and combinations thereof.

32. (canceled)

33. (canceled)

34. (canceled)

35. (canceled)

36. (canceled)

37. (canceled)

38. (canceled)

39. (canceled)

40. (canceled)

41. (canceled)

42. (canceled)

43. (canceled)

44. (canceled)

45. (canceled)

46. (canceled)

47. (canceled)

48. (canceled)

49. (canceled)

50. (canceled)

51. (canceled)

52. (canceled)

53. (canceled)

54. (canceled)

55. (canceled)

56. (canceled)

57. (canceled)

58. (canceled)

59. (canceled)

60. (canceled)

61. (canceled)

62. (canceled)

63. (canceled)

64. (canceled)

65. (canceled)

66. (canceled)

67. (canceled)

68. (canceled)

69. (canceled)

70. (canceled)

71. (canceled)

72. (canceled)

73. (canceled)

74. (canceled)

75. (canceled)

76. A method of separating molecules by their molecular weight or size
comprising:a. providing a plurality of molecules, wherein each molecule
comprises a polar moiety and a lipophilic moiety;b. placing at least a
portion of the plurality of molecules in a running buffer comprising a
drag-tag, wherein the drag-tag comprises a structure, and wherein the
structure comprises a surfactant, a polymer or combinations thereof;c.
applying an electric field to the running buffer for a period of time;d.
forming a lipophilic interaction between the lipophilic moiety and the
drag-tag for a portion of the period of time;e. terminating the
lipophilic interaction formed during step (d) during the period of
time;f. repeating steps (d) and (e) throughout at least a portion of the
period of time; andg. discontinuing the electric field.

[0004]The invention relates to transiently binding a drag-tag to a
molecule during a separation modality.

[0005]2. Description of Related Art

[0006]The ultimate success of a DNA sequencing methodology is determined
by both the resolving power of the technique itself and its sensitivity
to small differences in migrational velocity over a broad range of
sequencing fragment lengths. For direct comparison of sequencing
efficiency between various sequencing methodologies, it is often
convenient to cite the length of read ("LOR")/unit time, usually in the
number of called bases/day. Current capillary gel electrophoresis
("CGE")-based separation approaches have a LOR of approximately 500 to
600 bases with run times on the order of 2.5 hours. This would translate
into approximately 5,000 bases/day for a single capillary instrument. For
a 96 capillary array, this value approaches 500,000 bases/day, clearly
indicating the advantage of massively parallel separations. This LOR is
used as an indication of a technique's sequencing capacity. In CGE-based
techniques, the major obstacle to longer sequencing read lengths is
diffusion band broadening, a result of the long run times required to
avoid biased reptation.

[0007]End labeled free solution electrophoresis (ELFSE) is a DNA
separation modality capable of breaking the charge-to-friction ratio of
DNA in a gel- or polymer-free context. At the foundation of this
particular technique lies the notion that by appending a drag inducing
entity (i.e. a "drag-tag"), predominantly one with little or no charge,
the charge-to-friction symmetry of the DNA target can be broken, and the
resulting DNA fragment can be separated. Since DNA acts as a
free-draining coil, the electrophoretic mobility of DNA can roughly be
described by q/f, or its charge-to-friction ratio, where q is the net
charge on the molecule and f is the molecular friction coefficient. By
covalently attaching an uncharged drag-tag to the DNA target, the
friction induced by the pendant drag-tag moiety decreases the
electrophoretic mobility through an increase in the hydrodynamic friction
of the resulting complex. The drag-tag/DNA complex thereby possesses a
charge equivalent to the DNA itself, while the combined hydrodynamic
friction of the drag-tag/DNA complex is increased by the hydrodynamic
friction of the entire complex, including the drag-tag.

[0008]Since the inception of ELFSE methods, a number of obstacles remain
to be overcome for the successful application of this methodology. Since
ELFSE-based approaches separate through inherently different physical
mechanisms, biased reptation is not a concern, and substantially higher
electric field strengths (and the short run times that would result) are
achievable, representing an opportunity for improved separation
performance. Unfortunately, there are entirely different limitations with
ELFSE. The lack of sufficiently large, monodispersed polymeric drag-tags
currently limits the achievable LOR for ELFSE based approaches to around
125 bases.

SUMMARY OF THE INVENTION

[0009]Until now, limitations on the ability to produce large,
monodispersed drag-tags, has limited the applicability of methods such as
ELFSE. This problem is resolved by the present invention, which
transiently attaches drag-tags to the DNA fragment, as opposed to
covalently attaching polydispersed polymers thereto. The dynamic nature
of a drag-tag renders these separation modalities immune to the
aggressive band broadening effects of drag-tag polydispersity and it is
the transient nature of these interactions that offers the most distinct
advantage over traditional ELFSE based approaches.

[0010]Therefore, if monodispersion of drag-tags is not feasible, the
polydispersed drag-tags will randomly interact with a molecule.
Consequently, the net effect of random interactions between drag-tags of
different size eliminates the need for monodispersed drag-tags.

[0011]In some non-limiting embodiments, the invention generally relates to
the concept of transiently binding a drag-tag to a molecule during a
separation modality, such as electrophoresis. The separation modality
occurs in the presence of a running buffer. The molecule comprises at
least one polar moiety and at least one lipophilic moiety. The running
buffer comprises a drag-tag. The drag-tag comprises a structure. The
structure comprises a surfactant, a polymer or combinations thereof.
During electrophoresis, for example, the molecule moves by the
electrostatic force between the polar moiety and the electric field.
During at least a portion of this movement, the lipophilic moiety
hydrophobically interacts with the drag-tag. Thermal motion or an
electrostatic force eventually breaks the hydrophobic interaction,
thereby freeing the lipophilic moiety from the drag-tag and enabling the
lipophilic moiety to interact with a second, different drag-tag.

[0012]In some non-limiting embodiments, the invention comprises a running
buffer comprising a drag-tag. The drag-tag comprises a structure. The
structure can be selected from the group consisting of a liposome, a
micelle, a solid particle, a carbon nanotube, and an oil-in-water
emulsion. The oil-in-water emulsions can be micro-emulsions or
nano-emulsions. The structure comprises a surfactant, a polymer or a
combination thereof. In embodiments where the structure is a solid
particle or a carbon nanotube, the solid particle or carbon nanotube is
coated with the surfactant, the polymer or combinations thereof.

[0013]In some non-limiting embodiments, the present invention comprises a
molecule comprising at least one lipophilic moiety. In some embodiments,
the molecule comprises a plurality of lipophilic moieties. The molecule
comprises a nucleoside analog. The nucleoside analog comprises a sugar
moiety having a 5' end carbon and a 3' end carbon, and a lipophilic
moiety. The lipophilic moiety can be bonded to the 5' end, the 3' end or
elsewhere on the sugar moiety. The lipophilic moiety can be bonded to the
molecule by a linking group.

[0014]In some non-limiting embodiments, the molecule comprises a nucleic
acid analog. The nucleic acid analog comprises a sugar moiety having a 5'
end carbon and a 3' end carbon, and a lipophilic moiety. The lipophilic
moiety can be bonded to the 5' end, 3' end or elsewhere on the sugar
moiety.

[0015]In some non-limiting embodiments, the molecule comprises a
protein-detergent complex. The protein-detergent complex comprises at
least one protein and at least one lipophilic moiety bonded to at least a
portion of the protein.

[0016]In some non-limiting embodiments, the invention comprises a
lipophilic moiety comprising an alkyl group. The alkyl group can be
selected from the group consisting of an octyl, a nonyl, a decyl, an
undecyl, a dodecyl, a tridecyl, a tetradecyl, a pentadecyl, a hexadecyl,
a heptadecyl, an octadecyl, a nonadecyl, an icosyl, a henicosyl, a
docosyl, a tricosyl and a tetracosyl group. The lipophilic moiety further
comprises a first functional group attached to the alkyl group. The
lipophilic moiety further comprises a second functional group capable of
bonding to a molecule.

[0017]In some non-limiting embodiments, the invention comprises a method
of transiently attaching a drag-tag to a molecule. The method comprises
providing a molecule comprising a polar moiety and a lipophilic moiety.
The molecule is moved through the running buffer described above.

[0018]In some non-limiting embodiments, the invention comprises a method
of transiently attaching a drag-tag to a molecule. The method comprises
providing a molecule comprising a polar moiety and a lipophilic moiety.
The molecule is placed in the running buffer described above. An electric
field is applied to the running buffer for a period of time. A lipophilic
interaction is formed between the lipophilic moiety and the drag-tag for
a portion of the period of time. The lipophilic interaction is terminated
during the period of time. The formation and termination of the
lipophilic interaction are repeated through at least a portion of the
period of time. The electric field is discontinued.

[0019]In some non-limiting embodiments, the invention comprises a method
of separating molecules of different lengths. The method comprises
providing at least two molecules, each molecule having a different length
or size. Each molecule comprises a lipophilic moiety. The molecules are
separated by electrophoresis using the running buffer described above.

[0020]In some non-limiting embodiments, the invention comprises a method
of separating molecules of different lengths or size. The method
comprises forming a hydrophobic interaction between a lipophilic moiety
bonded to the molecule and a drag-tag. This hydrophobic interaction
occurs during a separating modality, such as electrophoresis. The
hydrophobic interaction is discontinued during the separation modality.
The formation and discontinuation of the hydrophobic interaction is
repeated throughout at least a portion of the separation modality. The
molecules separate at least two different distances.

[0021]In some non-limiting embodiments, the invention comprises a method
of measuring a hydrodynamic radius of a drag-tag. The method comprises
providing a plurality of molecules. The plurality of molecules comprises
a polar moiety and a lipophilic moiety, and each molecule comprises an
approximately equal molecular weight. The plurality of molecules is moved
a distance in the running buffer described above. Consequently, the
distance can be used to determine the hydrodynamic radius of the
drag-tag. The molecule can be a nucleic acid analog(s) of known size(s)
comprising at least one lipophilic moiety.

[0022]In some non-limiting embodiments, the invention comprises a method
of separating a plurality of drag-tags having different hydrodynamic
radiuses. The method comprises providing a plurality of molecules. The
molecules comprise a polar moiety and a lipophilic moiety, and have
approximately an equal molecular weight. The molecules also are capable
of binding tightly to the drag-tags described above, wherein the
drag-tags have different hydrodynamic radiuses. The molecules are
separated in the running buffer described above, and consequently, the
drag-tags are also separated by their hydrodynamic radius.

BRIEF DESCRIPTION OF THE DRAWING(S)

[0023]FIG. 1 is an illustration of a DNA molecule having a hydrodynamic
drag-tag attached thereto.

[0024]FIG. 2 is a chart demonstrating the impact of transient interactions
on drag-tag polydispersity.

[0025]FIG. 3 is a schematic representation of the addition of a long chain
alcohol to a chlorophosphoramidite and the subsequent attachment to a
support bound oligonucleotide.

[0026]FIG. 4 is a schematic representation of a post-synthetic
modification of a commercially synthesized 5' phosphorylated DNA
oligonucleotide resulting in a phosphoramidate linkage between the
oligonucleotide and a long chain primary amine.

[0027]FIG. 5 is a schematic representation of a post-synthetic
modification of a commercially synthesized 5' amine-labeled DNA
oligonucleotide resulting in an amide linkage between the oligonucleotide
and a long chain fatty acid.

[0038]FIG. 16 is a graph illustrating data from sequencing of M13 mp18
ssDNA template with a C18-aDNA primer (C18-M13 (-47)).

DETAILED DESCRIPTION OF THE INVENTION

[0039]Other than in the operating examples, or where otherwise indicated,
all numbers expressing quantities of ingredients, thermal conditions, and
so forth, used in the specification and claims are to be understood as
being modified in all instances by the term "about". Accordingly, unless
indicated to the contrary, the numerical parameters set forth in the
following specification and attached claims are approximations that may
vary depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to limit the
application of the doctrine of equivalents to the scope of the claims,
each numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary rounding
techniques.

[0040]Notwithstanding that the numerical ranges and parameters setting
forth the broad scope of the invention are approximations, the numerical
values set forth in the specific examples are reported as precisely as
possible. Any numerical value, however, inherently contains certain
errors necessarily resulting from the standard deviation found in their
respective testing measurements. Furthermore, when numerical ranges of
varying scope are set forth herein, it is contemplated that any
combination of these values, inclusive of the recited values, may be
used.

[0041]Also, it should be understood that any numerical range recited
herein is intended to include all sub-ranges subsumed therein. For
example, a range of "1 to 10" is intended to include all sub-ranges
between and including the recited minimum value of 1 and the recited
maximum value of 10, that is, having a minimum value equal to or greater
than 1 and a maximum value of equal to or less than 10.

[0043]This invention is captured by several embodiments. One embodiment of
the invention is a method of transiently binding a drag-tag to a
molecule. Another embodiment of the invention is a molecule comprising a
lipophilic moiety. Another embodiment of the invention is a lipophilic
moiety. Another embodiment of the invention is a method of measuring a
hydrodynamic radius of a drag-tag. Another embodiment of the invention is
a method of separating a plurality of drag-tag by their hydrodynamic
radiuses. Another embodiment of the invention is a running buffer.

[0044]Transiently Attaching a Drag-Tag to a Molecule

[0045]A non-limiting embodiment of the invention is a method of
transiently binding a drag-tag to a molecule. The method comprises
providing a molecule. The molecule comprises at least one polar moiety
and at least one lipophilic moiety. The molecule is moved through a
running buffer. The running buffer comprises a drag-tag.

[0046]Another non-limiting embodiment of the invention is a method of
transiently binding a drag-tag to a molecule. The method comprises
providing a molecule. The molecule comprises at least one polar moiety
and at least one lipophilic moiety. The molecule is placed in a running
buffer comprising a drag-tag. An electric field is applied to the running
buffer for a period of time. During at least a portion of the period of
time, a hydrophobic interaction is formed between the lipophilic moiety
and the drag-tag. The hydrophobic interaction is terminated. The
formation and termination of the hydrophobic interaction is repeated for
at least a portion of the period of time. The electric field is
discontinued.

[0047]Another non-limiting embodiment of the invention is a method of
separating molecules. The method comprises providing a plurality of
molecules having at least two different lengths. Each molecule comprises
at least one polar moiety and at least one lipophilic moiety. The
molecules are separated by a separation modality in a running buffer. The
running buffer comprises a drag-tag.

[0048]Another non-limiting embodiment of the invention is a method of
separating molecules by their molecular weight or size. The method
comprises providing a plurality of molecules. Each molecule comprises at
least one polar moiety and at least one lipophilic moiety. At least a
portion of the molecules is placed in a running buffer comprising a
drag-tag. An electric field is applied to the running buffer for a period
of time. During at least a portion of the period of time, a hydrophobic
interaction is formed between the lipophilic moiety and the drag-tag. The
hydrophobic interaction is terminated. The formation and termination of
the hydrophobic interaction is repeated for at least a portion of the
period of time. The electric field is discontinued.

[0049]One of ordinary skill in the art would recognize a polar moiety of a
molecule that would be useful in this embodiment. For example, if the
molecule is a nucleic acid analog, the polar moiety would include a
phosphate group found on the backbone of the nucleic acid.

[0050]In another example, if the molecule is a protein-detergent complex,
the polar moiety would include the amino acid(s) that provide a net
charge to the protein/detergent complex at a particular pH.

[0051]In some non-limiting embodiments, the lipophilic moiety comprises an
alkyl group. The alkyl group can be linear and/or saturated. It can
comprise at least about 8 carbon atoms, at least about 12 carbon atoms,
no more than about 24 carbon atoms or no more than about 18 carbon atoms.
For example, the alkyl group can be selected from the group consisting of
an octyl, a nonyl, a decyl, an undecyl, a dodecyl, a tridecyl, a
tetradecyl, a pentadecyl, a hexadecyl, a heptadecyl, an octadecyl, a
nonadecyl, an icosyl, a henicosyl, a docosyl, a tricosyl and a tetracosyl
group. The lipophilic moieties can also comprise fluorinated hydrocarbons
or fluorocarbons. The fluorocarbons can be selected from the group
consisting of C8 F15, C10 F19 and C12 F23
and C6 F13. In another embodiment, at least a portion of the
plurality of molecules comprises a lipophilic moiety. Each lipophilic
moiety can comprise approximately an equal number of carbon atoms.

[0052]The lipophilic moiety can comprise a functional group. In some
non-limiting embodiments, the functional group is a chromophore. In other
non-limiting embodiments, the functional group is a fluorophore. In other
non-limiting embodiments, the functional group is boron-dipyrromethene.
For example, the lipophilic moiety can comprise a Bodipy fluorophore. Due
to the uncharged-hydrophobic nature of the Bodipy fluorophore, fatty acid
derivatives of Bodipy are well suited for the conjugation to DNA
oligonucleotides to create a fluorescently labeled aDNA. The chemical
structure of the numerous aliphatic groups successfully coupled to a DNA
oligonucleotide may be found in FIG. 6. In other non-limiting
embodiments, the functional group can comprise a radioactive atom, such a
32P or 33P.

[0055]The molecules can be any molecule that one skilled in the art would
recognize as capable of being separated by a separation modality, such as
electrophoresis. In some non-limiting embodiments of the invention, the
molecule comprises a nucleic acid analog. The nucleic acid analog
comprises at least one lipophilic moiety. In these embodiments, the polar
moiety is the phosphate group in the backbone of the nucleic acid analog.
The lipophilic moiety is an alkyl group that is bonded to the nucleic
acid. In some non-limiting embodiments, the alkyl group is covalently
bonded to the sugar moiety of the nucleic acid. In other non-limiting
embodiments, the alkyl group is covalently bonded at the 3' end carbon or
the 5' end carbon of the sugar moiety.

[0056]In another non-limiting embodiment of the invention, the molecule
comprises a protein-detergent complex. The protein-detergent complex
comprises at least one amino acid. The amino acid comprises a moiety that
is the polar moiety. The protein-detergent complex further comprises at
least one lipophilic moiety. The lipophilic moiety can be a detergent.
The detergent can be selected from the group consisting of sodium dodecyl
sulfate, sodium lauryl sulfate and combinations thereof. The
protein-detergent complex can further comprise a protein denaturing
agent. Non-limiting examples of a protein denaturing agents include urea
and guanidinium hydrochloride. The lipophilic moiety forms a bond with
the amino acid. The bond can be an ionic bond.

[0057]A certain embodiment of the invention is a method of separating a
plurality of nucleic acids having at least two different lengths. This
embodiment comprises providing at least two nucleic acids having
different lengths. Each nucleic acid comprises a lipophilic moiety as
described above. The nucleic acids can be separated by thermal motion or
electrophoresis in a running buffer. The running buffer comprises a
drag-tag.

[0058]Another embodiment of the invention is a method of separating
nucleic acids of different lengths. This embodiment comprises forming a
hydrophobic interaction between a lipophilic moiety bonded to a nucleic
acid and a drag-tag during a separation modality. The hydrophobic
interaction is discontinued during the separation modality. Optionally,
the forming step and the discontinuing step are repeated.

[0059]In certain non-limiting embodiments, the lipophilic moiety can
hydrophobically interact with a first drag-tag during a separation
modality. The hydrophobic interaction is discontinued during the
separation modality. Thereafter, the lipophilic moiety can
hydrophobically interact with a second drag-tag tag during the separation
modality. The hydrophobic interaction between the lipophilic moiety and
the second drag-tag is discontinued. The formation and discontinuation of
hydrophobic interactions can be repeated between numerous random
drag-tags and the same lipophilic moiety.

[0060]The separation modality can be selected from the group consisting of
end-labeled free solution electrophoresis (ELFSE), micellular
electrokinetic chromatography, microemulsion electrokinetic
chromatography, liposome electrokinetic chromatography, and capillary
electrophoresis. Certain embodiments of the invention are devices
comprising a power-source. The power-source is capable of generating
electric fields of at least 100 V/cm, at least 500 V/cm, at least 1,000
V/cm, or at least 10,000 V/cm.

[0061]For convenience, the present invention is illustrated by the
non-limiting ELFSE separation modality of DNA in a micelle-containing
running buffer. A means to disrupt the insensitivity of the mobility to
nucleic acid length is required to practice the invention. This can be
accomplished by altering the hydrodynamic drag that the nucleic acid
molecule possesses by separating the nucleic acid in a separation
modality. Prior to this invention, it was assumed that the drag-tag had
to be covalently bonded to the DNA fragment. Using this model, any
polydispersity present in the size and/or shape of the drag-tag
population would translate into deviations imparted to various DNA
fragments of equal length, and subsequent loss of resolution in DNA
sequencing. It was determined that a polydispersity value of 1.00001 was
sufficient to virtually eliminate these deviations in DNA sequencing.

[0062]The influence that polydispersity has on the resolution of nucleic
acid separation modality, such as modalities used for DNA sequencing, is
most easily addressed by assuming that the drag-tag is an uncharged
polymer consisting of Mu identical, uncharged monomer units.
Assuming that each monomer unit has a molecular weight defined as
fwu, the number average molecular weight, Mn, of the polymer is
Mn=Mufwu. Provided that the polydispersity index (PDI) of
the polymer is known, (PDI=Mw/Mn), and that the average
molecular weight, Mw, of the polymer follows a normal distribution,
the standard deviation of the molecular weight is
σ2Mn=M2n(PDI-1). The error introduced through
polydispersity is expected to increase the temporal variance according to
σt--.sub.Total2=σt--Diff2+-
σt--.sub.Poly2, where σt poly2 poly
is the temporal variance caused by polydispersity in the drag-tag
molecular weight, calculated through propagation of error from
σ2Mn calculated as

[0063]According to this equation, to bring the length of read (LOR) up to
a value of 130, the PDI would have to be 1.00001. This has not heretofore
been accomplished using the currently existing separation modalities.

[0064]Use of a transiently attached drag-tag has the advantage that
increased sampling of a drag-tag during the separation results in
increased monodispersity in the degree of hydrodynamic drag imparted by
the tag. The hydrodynamic drag experienced by a particular DNA fragment
and its subsequent electrophoretic mobility is the average of numerous
interactions between different drag-tags. Provided that the number of
interactions is sufficiently large, each DNA fragment of a specific
length is expected to hydrophobically interact with an equivalent
distribution of drag-tag morphologies and each fragment's resultant
average electrophoretic mobility is expected to be equal. This does not
imply that each population of DNA fragment lengths will experience the
same number of interactions, since fragments moving more slowly would
spend a longer time within the capillary. However, each DNA fragment of a
specific length is expected to experience an equivalent number of
interactions, and as a result, should hydrophobically interact with an
approximately equivalent distribution of drag-tag morphologies.

[0065]The polydispersity resulting from x transient interactions,
PDIX with a drag-tag possessing a polydispersity of PDI is given by

PDI x = σ Mnx 2 M n 2 + 1 , ##EQU00003##

where σMnx is the standard deviation in the population
generated by sampling Mn over x interactions. Provided Mn is
normally distributed, σMnx is equal to the standard error, SE,

SE = σ Mnx = σ Mn x . ##EQU00004##

For a polymer drag-tag with a PDI=1.01, PDI1000=1.00001, indicating
that through the use of a transiently attached drag-tag, ELFSE-based
separations for DNA sequencing are indeed feasible. An example of the
probability distribution calculated from the predicted polydispersity for
various numbers of interactions is shown in FIG. 2. Each figure
represents the probability function distribution for x transient
interactions. In this figure, the probability distribution is determined
by the following formula:

f ( x ) = 1 SE 2 π exp ( - ( x - x ) 2
2 SE 2 ) . ##EQU00005##

[0066]It is readily observable that a sufficient number of interactions
with even a moderately polydispersed plurality of drag-tags has the
ability to render the effective drag induced by the tag virtually
monodispersed. For applications in DNA sequencing, the size resolution
factor, R(M), need only be adjusted slightly to account for transient
interactions:

Adopting identical experimental conditions as above and assuming PDI=1.01,
a single, permanent interaction, predicts LOR=4 bases. For x=1000
interactions, the predicted LOR=131 bases. If the number of interactions
approaches 1×106, LOR=457 bases, only three bases short of the
value predicted for a perfectly monodispersed polymer drag-tag.

[0067]Implementing the notion of transiently attaching a polymer drag-tag
is not immediately straightforward. If, for example, one were to rely on
a physical interaction between an uncharged polymer and a DNA molecule,
it is unlikely that there would be exactly one site of interaction on the
DNA molecule at any given instance. If one were to rely on a specific
interaction such as that afforded by the receptor-ligand pair
streptavidin and biotin, it may be difficult to tune the binding strength
such that there are a sufficient number of interactions to appreciably
lower the effective polydispersity of the drag-tag. To circumvent this
issue, the present inventors have determined that a lipophilic moiety can
be appended to a molecule, for example a nucleic acid. Although the
lipophilic moiety is not expected to impart a significant amount of drag
to the DNA fragment, the transient interaction of that group with a
large, uncharged surfactant micelle is believed to behave in an
equivalent fashion to the polymeric drag-tag discussed above. Provided
that the micelle is sufficiently large, with an equivalent drag of
˜150 bases, and the lifetime of a DNA/micelle interaction is
sufficiently short, the increase in effective monodispersity afforded by
the transient nature of the interaction would lead to competitive DNA
sequencing technologies using ELFSE.

[0068]The polyanionic character of nucleic acids renders them largely
hydrophilic, and therefore limits its interaction with a micellular
subphase. As a consequence, micelles have not been used for the
separation of intact oligonucleotides. The use of hydrophobically
modified nucleic acids promotes this DNA/micelle interaction, and is
believed to be at least partially responsible for the separations
achieved throughout this invention.

[0069]A nucleic acid can only interact with one of the two phases (aqueous
phase or the micellular phase) at any instant and the interaction process
can be modeled as an equilibrium reaction DNAaq+MicDNAm. The
propensity for a nucleic acid to interact with either phase can be
predicted by

where an equilibrium constant or partition coefficient, K, is defined as:

K ≡ [ DNA m ] [ DNA aq ] [ Mic ] ##EQU00008##

where [DNAm] and [DNAaq] are the concentrations of the DNA
fragment in the micellular and aqueous phases respectively, and [Mic] is
the concentration of micelles. These equations give an indication of the
propensity of a DNA fragment to reside in either of the two phases, the
aqueous phase or the micelle phase, provided the partition coefficient,
K, is known.

[0070]The partition coefficient serves as an efficient measure of the
interaction process. The poly-anionic character of DNA renders it
sufficiently hydrophilic so that interaction with the micellular phase is
highly unlikely. To promote micelle/DNA interaction, a hydrophobic group
is attached to the DNA fragment in the form of a simple aliphatic carbon
chain ranging in length from 8-24 carbons, 12-24 carbons, 8-18 carbons or
12-18 carbons. It is believed that a carbon tail ranging as short as 8
carbons, and as long as 24 carbons would be effective in various cases.
Upon addition of a substantially hydrophobic group, such as the C1-8
alkane chain, the partition coefficient, K, is approximately 1500,
indicating the fraction of time the modified DNA spends attached to the
micellular phase (fmic) is nearly one.

[0071]Once the extent of interaction with a micellular phase is known, the
effective mobility, μeff, of the DNA population can be
calculated. This is accomplished by assuming that the DNA's effective
mobility is simply a weighted average, weighted by the fractional
micellular interaction; of the intrinsic mobility of the fragment,
μDNA; and the mobility of the micelle, μmic; according to
μeff=faqμDNA+fmicμmic. Thus, the
effective mobility can be calculated according to

[0072]Although an uncharged, non-ionic surfactant micelle,
(μmic=0), was used for the majority of separations described
herein, this quantity is more appropriately defined as the mobility of a
micelle while it is interacting with the DNA, rather than the micelle
mobility alone. Since the DNA has a substantial electrophoretic mobility,
the mobility of the micelle while interacting with the DNA fragment does
not reflect the intrinsic mobility of the micelle itself. This
micelle/DNA complex mobility is governed by physics similar to a
covalently attached drag-tag.

[0073]Moreover, charged surfactants may be used, so long as the mobility
of the drag-tag differs from the mobility of the molecule. For example,
charged micelles can be used in free-solution separation of alkylated DNA
analogs, so long as the DNA analogs have a greater electrophoretic
mobility than the micelles.

[0074]There are a few considerations that need to be taken into account to
permit an accurate prediction of the effective or average electrophoretic
mobility of the DNA fragment. One of the main considerations is the fact
that the presence of micelles within the running buffer increases its
viscosity. Since electrophoretic mobility scales as 1/η, this has the
impact of decreasing the experimentally measured electrophoretic mobility
of the hydrophobically tagged DNA molecule. However, if every DNA
fragment present in the sample is labeled with the hydrophobic aliphatic
tail, the intrinsic mobility of the DNA fragment in the absence of a
micelle is not experimentally observable. This is due to the fact that
the only measurable indication is the effective, or average,
electrophoretic mobility, and as a consequence, neither μDNA nor
μmic is measured directly. Corrections for viscosity are taken
into account by the following equations:

directly relates the experimentally measurable effective or average
mobility of the hydrophobically labeled DNA to the total concentration of
micelles in the running buffer through the partition coefficient K, and
the micelle mobility μomic. In an effort to determine these
two coefficients,

[0075]By conducting a series of electrophoretic separations, each at a
different surfactant concentration, the effective mobility of the DNA
fragment can be determined. When combined with knowledge of the viscosity
constant and the intrinsic mobility of the DNA fragment, measured in the
absence of surfactant micelles, the above equation can readily be solved
by linear regression.

[0076]The aliphatic tail is likely to have no detectable impact on the
electrophoretic mobility of the nucleic acid analog. This is a result of
the fact that the amount of drag associated with 24 carbons is not
sufficient to alter the electrophoretic mobility of the substantially
larger DNA fragment. This assumption is valid for all but the shortest
DNA fragments. The remainder of the separation can be described quite
simply. While a hydrophobically modified DNA target is interacting with a
micelle of size α, it would possess an electrophoretic mobility
defined by μomic. The value of μomic is
determined by the combined drag of the uncharged surfactant micelle, the
intrinsic mobility of the DNA fragment, μoDNA=μo,
and the length of the DNA fragment, Mc, according to

μ = μ ∘ M c M c + α .
##EQU00016##

For the fraction of the separation that the DNA fragment is not
interacting with the micellular phase, the DNA would migrate at its
free-solution electrophoretic mobility, μoDNA. Thus, the
effective mobility of alkylated DNA analogs can be calculated according
to

[0077]The size resolution factor R(M) can accurately evaluate the impact
that of the micellular interaction on the resolution of DNA fragments.
The impact that polydispersity of micelles is expected to be negligible
provided there are a sufficient number of interactions between the DNA
fragment and the micellular drag-tag. This can be confirmed by
calculating R(M) according to the following equation:

[0078]Applying hypothetical experimental conditions where E=333 V/cm,
lD=34 cm, D1=3.2×10-6 cm2/s,
μoDNA=-1.95×104 cm2/Vs and α=24, the
predicted LOR is 127 bases. At a micelle concentration of 0.1 mM, with
K=1000 mM-1 and Cvisc=0.5 mM-1, the predicted LOR is 125
bases. For α=150 and E=1000 V/cm, a transient attachment of a
surfactant micelle predicts a LOR=447 bases, only 13 bases below the 460
bases, which is the predicted LOR for a covalently attached monodispersed
drag-tag. This deviation is due in large part to the contribution that
the increased solution viscosity has on the separation, and if viscosity
effects are assumed to be negligible, the LOR would be 455 bases. This is
readily explainable by the fact that, all, or almost all of DNA fragments
are expected to be interacting with a surfactant micelle at any given
instant.

[0079]Nucleoside Analogs and Nucleic Acid Analogs

[0080]Another embodiment of the invention is a nucleoside analog. The
nucleoside analog comprises a nucleoside bonded to a lipophilic moiety.
The nucleoside is a glycosylamine comprising a sugar moiety selected from
the group consisting of a ribose or a deoxyribose, and a nucleobase
attached to the sugar moiety. The nucleobase can be selected from the
group consisting of adenosine, cytidine, guanosine, thymidine and
uridine.

[0081]The sugar moiety comprises a 3' end carbon and a 5' end carbon. The
lipophilic moiety can be bonded or directly bonded to a sugar. In one
non-limiting embodiment, the lipophilic moiety is bonded or directly
bonded to the 3' end carbon. In another non-limiting embodiment, the
lipophilic moiety is bonded or directly bonded to the 5' end carbon. In
one embodiment, the nucleoside analog comprises a plurality of lipophilic
moieties. The lipophilic moiety can be bonded to the nucleoside by a
linking group. The linking group is a group that bonds the lipophilic
moiety and the nucleoside. One skilled in the art would recognize groups
that bond between lipophilic moieties and nucleosides. Non-limiting
examples of linking groups include an amide, a phosphoramidite bond and a
phosphodiester bond.

[0082]The lipophilic moiety can comprise an alkyl group. The alkyl group
can be linear and/or saturated. It can comprise at least about 8 carbons,
at least about 12 carbons, no more than about 24 carbons or no more than
about 18 carbons. The alkyl group can be selected from the group
consisting of an octyl, a nonyl, a decyl, an undecyl, a dodecyl, a
tridecyl, a tetradecyl, a pentadecyl, a hexadecyl, a heptadecyl, an
octadecyl, a nonadecyl, an icosyl, a henicosyl, a docosyl, a tricosyl and
a tetracosyl group. The lipophilic moieties can also comprise fluorinated
hydrocarbons or fluorocarbons. The fluorocarbons can be selected from the
group consisting of C8 F15, C10 F19 and C12
F23 and C6 F13.

[0083]The lipophilic moiety can optionally comprise a functional group. In
some non-limiting embodiments, the functional group is a chromophore. In
other non-limiting embodiments, the functional group is a fluorophore. In
other non-limiting embodiments, the functional group is
boron-dipyrromethene. In other non-limiting embodiments, the functional
group comprises a radioactive atom. For example, the functional group can
comprise a radioactive phosphorus, such as 32P or 33P.

[0084]Another embodiment of the invention is a nucleic acid analog. The
nucleic acid analog comprises at least one nucleoside analog described
above.

[0085]In some embodiments, the present invention comprises a primer for a
polymerase chain reaction comprising the nucleic acid analog.

[0086]Bonding the lipophilic moiety to the 5' end or the 3' end means
bonding the lipophilic moiety to the 5' carbon or 3' carbon, or bonding
the lipophilic moiety to a 5' nucleoside or a 3' nucleoside. Bonding the
lipophilic moiety to the nucleoside, for example the 5' nucleoside or the
3' nucleoside, can occur anywhere on the nucleoside.

[0087]Lipophilic Moiety

[0088]Some embodiments of the invention comprise a lipophilic moiety. The
lipophilic moiety comprises an alkyl group. The alkyl group can be linear
and/or saturated. It can comprise at least about 8 carbons, at least
about 12 carbons, no more than about 24 carbons or no more than about 18
carbons. The alkyl group may be selected from the group consisting of an
octyl, a nonyl, a decyl, an undecyl, a dodecyl, a tridecyl, a tetradecyl,
a pentadecyl, a hexadecyl, a heptadecyl, an octadecyl, a nonadecyl, an
icosyl, a henicosyl, a docosyl, a tricosyl and a tetracosyl group. The
lipophilic moieties can also comprise fluorinated hydrocarbons or
fluorocarbons. The fluorocarbons can be selected from the group
consisting of C8 F15, C10 F19 and C12 F23
and C6 F13.

[0089]The lipophilic moiety comprises a first functional group. The first
functional group can be useful in detecting a molecule. The method of
detection can be visual or by radiograph. In some non-limiting
embodiments, the functional group is a chromophore. In other non-limiting
embodiments, the functional group is a fluorophore. In other non-limiting
embodiments, the functional group is boron-dipyrromethene. In other
non-limiting embodiments, the functional group comprises a radioactive
atom.

[0090]The lipophilic moieties comprise a second functional group. The
second functional group is useful in attaching the lipophilic moieties to
a molecule. By way of example, the second functional group can be
selected from the group consisting of an alcohol, an amine and a
carboxylic acid.

[0091]Measuring a Hydrodynamic Radius of a Drag-Tag

[0092]In some embodiments, the invention is a method of measuring a
hydrodynamic radius of a drag-tag. The method comprises providing a
plurality of molecules. Each molecule comprises a polar moiety, and a
lipophilic moiety. The molecules comprise an approximately equal
molecular weight, or an equal molecular weight. The molecules can also
comprise an approximately equal charge, or an equal charge. For example,
if the molecule comprises a plurality of nucleic acid analogs as
described above, at least a portion of the plurality of the nucleic acid
analogs would have an approximately equal molecular weight, or an equal
molecular weight. This can be achieved by providing a portion of nucleic
acid analogs having an approximately equal number of nucleotides, or an
equal number of nucleotides. The nucleic acid analogs of this example
would likewise have an approximately equal charge, or an equal charge
because the net charge on a nucleic acid is approximately equal or equal.

[0093]The plurality of molecules is moved by a separation modality a
distance in a running buffer comprising the drag-tag. The drag-tag
comprises a structure having an unknown hydrodynamic radius. The
structure comprises a surfactant, a polymer or combinations thereof. The
lipophilic moieties on the drag-tags hydrophobically interact with the
molecule during at least a portion of the separation modality.
Consequently, the distance that the molecules move is related to the
hydrodynamic radius of the drag-tag. From this distance, the hydrodynamic
radius of the drag-tag can be determined. Generally, drag-tags in an
aqueous suspension with hydrodynamic radius between 1 nm and 1,000 nm can
be assayed using the method.

[0094]Separating Drag-Tags Based on Their Hydrodynamic Radius.

[0095]In some embodiments, the invention is a method of separating a
plurality of drag-tags having different hydrodynamic radiuses. The method
comprises providing a plurality of molecules. The molecules comprise a
polar moiety and a lipophilic moiety. The molecules comprise an
approximately equal molecular weight, or an equal molecular weight. The
molecules can also comprise an approximately equal charge, or an equal
charge. The molecule or lipophilic moiety further comprises the ability
to tightly bind to the drag-tag. The molecules are separated in a running
buffer comprising the drag-tags. The drag-tags comprise a structure
having different hydrodynamic radiuses. The structure comprises a
surfactant, polymer or combination thereof; as discussed above. As a
consequence of the tight bonding between the molecule or lipophilic
moiety and the drag-tags, the drag-tags are moved with the molecules a
distance dependent upon the hydrodynamic radius of each individual
drag-tag. Thus, the drag-tags would be separated by their hydrodynamic
radius. Generally, drag-tags in an aqueous suspension with hydrodynamic
radius between 1 nm and 1,000 nm can be assayed using the method.

[0099]Liposomes are formed by a variety of methods known to a person of
ordinary skill. Non-limiting examples of these methods include
sonication, pressure-filtration ("extrusion"), reverse-phase evaporation.
A non-limiting example of liposomes suitable for use in this invention is
a liposome composed of a 20:80 mixture of cholesterol and
dipalmitoylphosphotidylglycerol, extruded to achieve an average size of
100 nm.

[0100]Micelles are formed by a variety of methods known to a person of
ordinary skill. The micelles can be formed by adding a sufficient
concentration of surfactant to reach the critical micelle concentration.
At the critical micelle concentration, micelles spontaneously form.
Micelles can form if the concentration of the surfactant is in the range
of 10-6 and 10-3 M, depending on the surfactant in question.

[0101]The solid particles are coated with surfactants. The surfactants are
applied to the solid particle by any method known to one skilled in the
art. For example, a solid particle can be coated with a surfactant by
liposome or vesicle fusion process where silica beads are incubated with
liposomes, or by direct adsorption of surfactant and/or an associating
polymer. Examples of solid particles include gold, silver, platinum,
silica, titania, cadmium selenide, cadmium sulfide, indium arsenide, and
indium phosphide.

[0102]The carbon nanotubes are coated with surfactants. The surfactants
can be applied to the carbon nanotubes by any method known to one skilled
in the art. For example, carbon nanotube bundles can be dispersed as
single nanotubes by sonication in the presence of an adsorbing
surfactant. These surfactants can include sodium dodecyl sulfate, sodium
dodecyl benzene sulfonate, or octylphenol ethylene oxide condensate (such
as Triton X-100).

[0103]Oil-in-water emulsions are formed by methods known to a person of
ordinary skill in the art. They can be formed by adding the surfactant
and oil to the running buffer, thereby forming a colloid. The
oil-in-water emulsions can be micro-emulsions or nano-emulsions. For
example, the emulsions can be comprised of 0.1% mineral oil in water with
0.25 mg/ml of the non-ionic surfactant C16E6 in the water phase. Such
emulsions can be prepared to form particles in the size range of 40-100
nm by thermal quenching and are called "nano-emulsions."

[0104]The running buffer can further comprise a buffering system. The
buffering system can be selected from the group consisting of
tris(hydroxymethyl)aminomethane ("Tris") acetate, Tris HCl,
Tris-2-(N-morpholino)ethanesulfonic ac (MES), phosphate buffered saline,
Tris-acetate-EDTA (TAE) buffer, sodium chloride, and combination(s)
thereof. In some embodiments, the running buffer further comprises 10 mM
of Triton X-100 in 50 mM Tris-MES at pH 8.0.

[0105]In some embodiments, the running buffer comprises a non-ionic
drag-tag. The non-ionic drag-tag can be dispersed in a liquid colloid.
The non-ionic drag-tag can comprise a structure, wherein the structure is
a micelle.

[0106]Phosphoramidite Synthesis of a Nucleic Acid Analog

[0107]To construct a nucleoside analog or a nucleic acid analog, a
lipophilic moiety can be appended to a nucleoside or nucleic acid, for
example at the 5' end of a DNA molecule, creating an alkylated nucleic
acid or aDNA. The use of organic solvent systems, necessitated by the
water-insolubility of the aliphatic group, is generally incompatible with
the otherwise organic-insoluble oligonucleotide.

[0108]One example of synthesizing a hydrophobically-labeled nucleic acid
is by phosphoramidite synthesis. Since oligonucleotide synthesis
reactions typically take place in organic solvents, it would be ideal to
perform the alkylation step on the solid support, alleviating the
difficulty of finding a solvent system suitable for the coupling of the
hydrophobic group. The general process behind doing so is to begin with
the 2-cyanoethyl N,N' diisopropyl-chlorophosphoramidite molecule (FIG.
3). The highly reactive chloro-functional group of the phosphoramidite is
substituted with a more stable long chain alcohol, for example
octadecanol. This results in a Cis-labeled phosphoramidite that can
later be utilized in the solid phase synthesis reaction using an
automated oligonucleotide synthesizer. The phosphoramidite molecule was
designed such that the aliphatic group on the final synthesized
oligonucleotide would be linked through a phosphodiester bond and as a
result, the phosphoramidite lacks the chemical functionality for the
addition of subsequent phosphoramidites. This has the consequence that
the alkylation step must be the final step in the extension of the
oligonucleotide on the solid support. Since the oligonucleotide is
attached to the solid support at the 3' end of the DNA molecule, the
resulting aliphatic label resides at the 5' end of the synthesized
oligonucleotide. Following the deprotection of the oligonucleotide and
cleavage from the solid support, the aDNA oligonucleotide can be purified
using reverse phase HPLC.

Example 1

[0109]The phosphoramidite synthesis method was performed as follows. 135
mg of octadecanol (0.5 mmol) was added to a round bottom flask followed
by 2 ml of dry DCM (dried over molecular sieves) and sonicated to speed
dissolution. 174 μl of Diisopropylethylamine (1 mmol) was added and
allowed to equilibrate for 5 minutes. 112 μl of 2-cyanoethyl N,N'
diisopropyl-chlorophosphoramidite (0.5 mmol) was added and the reaction
was stirred at room temperature. After approximately 1 hr, the reaction
was diluted with 13 ml of DCM and washed with 30 ml of saturated
NaHCO3. The organic layer was collected and dried over
Na2SO4 and the solvent was removed by rotary evaporation. The
product was purified using flash chromatography over silica gel with a
hexane/ethyl acetate/triethylamine (4:1:0.05) mobile phase. The fraction
containing the product, as judged by TLC, was collected and solvent was
removed using a rotary evaporator. The resulting phosphoramidite was then
used during the final (5') coupling during automated DNA oligonucleotide
synthesis resulting in a C18 labeled oligonucleotide.

[0110]Phosphoramidate Linkage between a Nucleic Acid and a Lipophilic
Moiety

[0111]Another method of alkylating a nucleic acid, illustrated in FIG. 4,
is a post-synthetic modification of a 5' phosphorylated DNA
oligonucleotide resulting in a phosphoramidate linkage between the
oligonucleotide and a long chain primary amine. 5' phosphorylated DNA
oligonucleotide is precipitated out of aqueous solution using the
cationic surfactant cetyl trimethyl ammonium bromide (CTAB) and the
resulting DNA-CTAB salt is readily soluble in organic solvent. This step
is crucial in the success of the post-synthetic modification since, as
discussed previously, the major obstacle to conventional conjugation
methods is the incompatibility of the aqueous insoluble aliphatic group
and the organic insoluble oligonucleotide. Upon solubilization of the DNA
oligonucleotide in organic solvent, the 5' phosphate group is activated
with a mixture of 2,2'-dipyridyldisulfide, triphenylphosphine and DMAP
resulting in an activated terminal phosphate susceptible to nucleophilic
substitution by a long chain primary amine. Following conjugation, the
DNA is precipitated out of the organic solution using acetone and the
oligonucleotide is purified by HPLC. The resulting linkage between the
oligonucleotide and the aliphatic group is a phosphoramidate linkage.

Example 2

[0112]The phosphoramidate linkage between the oligonucleotide and a long
chain primary amine was formed according to the following method. 50
μl of 2.5 mM 24 nt, 5'-phosphorylated DNA (125 nmol) in water was
added to a microcentrifuge tube. The DNA was precipitated using 3
μmoles of CTAB in water (1:1 molar ratio of CTAB to phosphate groups).
The resultant suspension was dried in a Speedvac and the resulting
DNA/CTAB salt was resuspended in 50 μl of 0.8 M 4-(dimethylamino)
pyridine (40 μmol) in DMSO dried over molecular sieves. 25 μl of
1.2 M triphenylphosphine (30 μmol) and 25 μl of 1.2 M
2,2'-dipyridyldisulfide (30 μmol) was added and allowed to equilibrate
at room temperature for 15 minutes. 3 μl of TEAA (to prevent
protonation of the amine) and 100 μl of 0.125 M of octadecaneamine
(12.5 μmol) in DMSO at 50° C., (to promote dissolution of
C18NH2 in DMSO) were added and the mixture was gently stirred
at 50° C. for 30 minutes. The reaction mixture is precipitated
with 1 ml of 2% lithium perchlorate in acetone. The suspension was
centrifuged at maximal velocity (16100 g's) in a microcentrifuge and the
supernatant was removed. The pellet was washed with acetone 3 times,
resuspended by sonication, and centrifuged again. The supernatant was
removed and the DNA oligonucleotide was suspended in 1 ml of 0.1 M TEAA
buffer pH 7.0. The labeled oligonucleotide was purified using reverse
phase HPLC, on a C18 column with a 1 ml/min linear gradient from
100% TEAA to 100% acetonitrile over 30 minutes. The unmodified
oligonucleotide elutes at ˜11 min while the C18 labeled
oligonucleotide elutes at ˜22 minutes. The individual fractions are
collected and lyophilized prior to use.

[0113]Absorbance of reverse-phase HPLC-purified C18-aDNA confirms
that the alkylation and recovery of this alkylation process was
successful. The precipitation process was effective at removing the
majority of conjugation reactants but any residual un-reacted DNA
oligonucleotide co-precipitates with the desired aDNA. To remove the
residual DNA reactant, reverse phase HPLC was conducted as with the
solid-phase system above. The results of the HPLC purification are shown
in FIG. 8. The chromatogram in FIG. 8(A) represents the trace
corresponding to the phosphorylated DNA reactant. FIG. 8(B) is the trace
from the alkylation reaction mixture following acetone precipitation in
the presence of 0.1% trifluoroacetic acid. FIG. 8(C) is the trace from
the same alkylation reaction in the absence of TFA. TFA is frequently
added to the mobile phase in reverse-phase HPLC as an ion pairing agent
to promote the interaction of positively-charged analytes with the
hydrophobic media. The separation was largely devoid of
positively-charged analytes, so TFA may be unnecessary here. In fact,
comparing FIG. 8(B) and FIG. 8(C) demonstrates that the presence of TFA
has deleterious effects on the amount of recovered alkylated DNA. The
presence of TFA in the mobile phase substantially lowers the solvent's pH
and that the phosphoramidate linkage between the aliphatic group and the
DNA oligonucleotide appears to be labile under acidic conditions.
Comparison of the peak areas in these two traces indicates that the
effective coupling efficiency following purification is 11% in the
presence of TFA, and 34% in its absence. In addition, subsequent analysis
following successful PCR extension of the aDNA fragment recovered from
FIG. 8(C) indicated a loss of hydrophobic character following repeated
heating cycles, which is evidence of the fragile nature of the
phosphoramidate linkage between the aliphatic group and the
oligonucleotide. The lack of thermal stability of the aDNA molecule is a
major concern for applications where PCR extension is desirable, but
nonetheless, this method was effective in the alkylation of commercially
supplied oligonucleotides and for applications at physiological pH and
ambient temperatures, this method could prove useful.

[0114]Amide Linkage between a Nucleic Acid and a Lipophilic Moiety

[0115]Another method of alkylating a nucleoside or nucleic acid, such as a
DNA, illustrated in FIG. 5, is a post-synthetic modification of a 5'
amine-labeled DNA oligonucleotide resulting in an amide linkage between
the oligonucleotide and a long chain fatty acid. This method uses a 5'
amine-labeled oligonucleotide. A long chain fatty acid is activated with
DPDS, PPh3 and DMAP and mixed with the DNA-CTAB salt as before. The
reaction conditions are similar to the method resulting in the
phosphoramidite synthesis method; the main difference is the site of
activation and the resulting linkage. In the phosphoramidite synthesis
method, the phosphate group of the DNA oligonucleotide is activated while
in the second method, the activation takes place on the fatty acid itself
prior to incubation with the amine-labeled DNA. The linkage resulting
from this approach is an amide bond and proved to be more stable under
PCR thermocycling and HPLC purification conditions than the
phosphoramidate bond.

Example 3

[0116]The amide linkage between the oligonucleotide and a long chain fatty
acid was formed according to the following method. 5 μl of 1 M
4-(dimethylamino) pyridine (5 μmol) in DMSO dried over molecular
sieves, was added to a 1.5 ml centrifuge tube containing 20 μl of 5 mM
fatty acid or Bodipy fatty acid analog (100 nmol) and 1 μA
triethylamine. 5 μl of 0.5 M triphenylphosphine (2.5 μmol) and 5
μl of 0.5 M 2,2'-dipyridyldisulfide (2.5 pimp was added and allowed to
equilibrate at room temperature for 5 minutes. Meanwhile, 10 nmol of 24
nt, 5' amino-modified DNA (Integrated DNA Technologies, Coralville, Iowa)
in water was added to a centrifuge tube. The DNA was precipitated by
adding 230 nmol of CTAB in water (1:1 molar ratio of CTAB to phosphate
groups). The suspension was dried by vacuum centrifugation and the
resulting DNA/CTAB salt was resuspended in the reaction mixture, and
allowed to react with vigorous agitation for 2 hours. The reaction
mixture was precipitated with 1 ml of 2% lithium perchlorate in acetone.
The suspension was centrifuged at maximum velocity (16100 g's) in a
microcentrifuge and the supernatant is removed. The pellet was washed
with acetone 3 times, resuspended by sonication, and centrifuged again.
The supernatant was removed again and the DNA oligonucleotide was
suspended in 25 μl of 0.1 M triethylammoniumacetate buffer pH 7.0. The
labeled oligonucleotide was purified using reverse phase HPLC, on a
C18 column with a 1 ml/min linear gradient from 100% TEAA to 100%
acetonitrile over 60 minutes. The unmodified oligonucleotide eluted at
˜17 minutes while the alkylated DNA oligonucleotide eluted at
˜31 minutes. The individual fractions were collected and dried,
either through lyophilization or vacuum centrifugation prior to use.

[0117]Absorbance of reverse-phase HPLC-purified C18-aDNA confirms
that the alkylation and recovery of this alkylation process was
successful. The DNA oligonucleotide was precipitated out of aqueous
solution with the cationic surfactant CTAB, so that it would be
compatible with the organic solvent system required for the dissolution
of the fatty acid aliphatic group. The results from HPLC purification
from this reaction method can be found in FIG. 9. This particular
purification is of a 24-base oligonucleotide conjugated to the
hydrophobic C12-Bodipy-Fl fluorophore. As before, FIG. 9(A)
represents the chromatogram of the C12-Bodipy-Fl fatty acid. The
small peak at 30 minutes was an impurity that was injected with the fatty
acid control and did not affect the subsequent purification of the aDNA,
represented in FIG. 9(B). Comparison of peak areas indicated a coupling
efficiency of 27%. There is a small population evident immediately after
the unlabeled DNA reactant that has the characteristic UV spectrum of a
nucleic acid. This may be a side product of the conjugation reaction
itself. Since it elutes at a migration time markedly different from the
alkylated DNA, it is of little consequence for the purification of the
aDNA. The presence of TFA in the mobile phase did not seem to impact the
efficiency of the coupling nor did prolonged exposure to elevated
temperature, a result of the substantially more stable amide linkage
between the aliphatic group and the oligonucleotide.

[0118]These methods rely on the precipitation of DNA from aqueous solution
followed by suspension of the resulting DNA/CTAB salt in DMSO. Once the
DNA was solubilized within the organic solvent system, activation of the
terminal phosphate with DPDS, PPh3 and DMAP permitted the covalent
attachment of various long-chain carboxylic acids to the oligonucleotide.
Following the conjugation reaction, the alkylated DNA was precipitated
out of DMSO using acetone and LiClO4.

Example 4

[0119]In order for a separation modality to be capable of performing
separations of DNA sequencing products, the physical mechanism
responsible for the separation must be compatible with the extension of
the primer by DNA polymerase. Since the enzymes typically used for DNA
sequencing are minor mutations of enzymes commonly employed in PCR, the
ability of PCR enzymes to extend aDNA primers translated well to DNA
sequencing applications. Tests using a nucleic acid primer comprising a
lipophilic moiety bonded to the 5' end confirmed that the alkylation of
the nucleic acid does not inhibit DNA polymerase from extending the
primer, or the primer annealing to the template.

[0120]UV melting curves were used to determine the melting temperature of
the alkylated DNA generated using solid phase synthesis. UV melts were
conducted in a Varian Cary 3 spectrophotometer bearing a peltier
controlled cell holder. The 34 nt synthetic aDNA oligonucleotide was
incubated with its commercially-synthesized 34 nt complement at a final
concentration of 1 μM each in 50 mM Tris MES, pH 8.0. The sample was
heated to 95° C. and held for 5 minutes to ensure complete
denaturation of the two strands. The temperature was then lowered to
15° C. at a rate of 1° C./min while the absorbance was read
at 260 nm. The sample was held at 15° C. for 5 minutes prior to a
1° C./min heating cycle back to 95° C.

[0121]Sanger-type cycle sequencing reactions were performed to confirm
that alkylated DNA primers are compatible with this method, commonly used
for the generation of extension products for DNA sequencing purposes. The
reactions were conducted in a SmartCycler (Cepheid, Sunnyvale, Calif.) at
a total volume of 20 μl. Cycle sequencing reactions based on the
Therminator DNA polymerase included the following: 1000 nM C12
Bodipy-Fl-aDNA or C18-aDNA primer, 0.05 mM dNTP mixture 0.1 mM chain
terminator (acyATP or Fluorescein-labeled ddGTP), 1× Thermo Pol
buffer, (New England Biolabs, Ipswich, Mass.), 100 ng/μl M13 mp18
single-stranded DNA template, (NEB), and 0.05 Units/μl Therminator DNA
polymerase (NEB). The thermocycling conditions were as follows: An
initial template denaturation step of 5 minutes at 95° C. was
conducted, followed by 55 cycles of 95° C. for 30 seconds,
55° C. for 30 seconds, and 72° C. for 120 seconds. A final
elongation step following thermal cycling was conducted for 5 minutes at
72° C. Following reaction, residual nucleotide triphosphates and
reaction buffer salts were removed using a Centri-Sep column. (Princeton
Separations, Princeton, N.J.). The resulting purification product was
either mixed with 2×TBE-Urea loading buffer (Promega) prior to
denaturing polyacrylamide gel electrophoresis, or loaded into the
capillary electrophoresis in distilled water.

[0122]Extension success was measured by polyacrylamide gel electrophoresis
(PAGE). Polyacrylamide gels were prepared by combining 40% acrylamide/bis
acrylamide (37.5:1) solution (BioRad Laboratories, Hercules, Calif.) in
10×TBE buffer (89 mM Tris base, 89 mM Boric acid, 20 mM EDTA pH
8.3) and DI water. The mixture was vortexed briefly and degassed in a
vacuum chamber for 15 minutes. Following degassing,
N,N,N',N'-Tetramethylethylenediamine and ammonium persulfate were added
to final concentrations of 0.5% v/v and 0.01% w/v respectively.
Typically, gels were cast to a final acrylamide concentration of 5% into
1 mM thick, 7.3 cm tall, 8 mM wide gels. Cast gels were loaded into a
Mini-Protean 3 vertical electrophoresis chamber (BioRad Labs) and a
mixture of the DNA to be analyzed and 6× loading dye (Promega) in
water were loaded into the gel. A voltage of 150-200 V was applied
(E≈20-27 V/cm) and typical run times were approximately 45
minutes. Following electrophoresis, the gel was stained with Ethidium
Bromide and visualized with a BioDocIt transilluminator (UVP, Upland,
Calif.). The lengths of the fragments in the ladder, in bp, are in the
column on the left. The contents of each of the 9 lanes are as follows:
1) PCR marker ladder, 2) 107 by unlabeled DNA, 3) 107 by
C12-Bodipy-Fl labeled aDNA, 4) 107 by unlabeled DNA, 5) 107 by
C12-Bodipy-Fl labeled aDNA, 6) 255 by unlabeled DNA, 7) 255 by
C12-Bodipy-Fl labeled aDNA, 8) 450 by unlabeled DNA, 9) 450 by
C12-Bodipy-Fl labeled aDNA. Staining was conducted with ethidium
bromide (FIG. 10).

[0123]The gels confirmed that the alkylated nucleic acid primers were
extended successfully (see FIG. 10). The first and most important feature
of this figure is that the addition of a hydrophobic group, (a
C12-Bodipy fatty acid tail) does not appreciably hinder the
polymerase's ability to extend the labeled primer. There is approximately
a 20% reduction in the band intensity for each of the alkylated PCR
products as judged by subsequent densitometric scans, indicating a slight
extension bias against the labeled primer. The PCR reaction was equally
specific for the labeled primer and the resultant band intensities are
indicative of a substantial degree of primer amplification.

[0124]Another important feature present in the above gel is the slight
shift between labeled and unlabeled PCR fragments. This is more prevalent
for the shortest PCR products, specifically the 107 by product found in
lanes 2 and 3. This is to be expected, owing to the fact that the
electrophoretic mobility shift induced by the aliphatic tail will have
diminishing impact on increasing lengths of DNA targets. This shift could
be explained by an increased hydrodynamic drag relevant to the naked DNA
itself, following a mechanism analogous to the ELFSE-based drag in free
solution. Namely, the tail is able to break the charge-to-friction ratio
of the DNA fragment while it is migrating through the pores of the
polyacrylamide. The drag associated with the relatively small lipophilic
moiety would not be substantial enough to induce an appreciable shift for
long PCR products, consistent with the above observation that there is
only a detectable shift for the shortest two product lengths. Secondly,
since the polyacrylamide gel matrix possesses a moderate degree of
hydrophobic character, the tag could be transiently associating with the
matrix itself. Since the matrix is stationary, any favorable interactions
that the PCR fragment has with the gel matrix would retard the migration
of the fragment, even if the interactions are weak. This would be
analogous to the drag induced by a drag-tag with an infinite a, and any
length dependence of the electrophoretic mobility shift caused by the
hydrophobic group must be a result of differential interaction with the
stationary gel matrix. It is unknown which of these two factors would
dominate but since the focus of this work is aimed at the separation of
DNA fragments in the absence of a polymer matrix, it will simply be left
as a curiosity. It does, however, suggest a new separation modality,
namely that while conducting a gel electrophoresis experiment, micelles
could be added to the running buffer to induce an even greater
electrophoretic mobility shift of the alkylated PCR fragments.

[0125]Although three successful methods were established for DNA
alkylation, the post-synthetic modification of a 5' amine labeled DNA
with a long chain fatty acid presented the greatest degree of sequence
reliability, ease of conjugation and alkylation stability. The
characterization of the conjugation involved ascertaining the degree of
hydrophobicity conferred through the alkylation in reverse-phase HPLC and
the subsequent extension of the alkylated primer in PCR.

[0127]The Therminator DNA polymerase was used to extend the C18
primer in a sequence dependent fashion along the single-stranded M13mp18
DNA template. Termination of sequencing fragments was accomplished
through the use of a fluorescein-labeled ddGTP (ddGTP-Fl) chain
terminator. As a first pass, the generation of sequencing fragments from
a single chain terminator was investigated for proof-of-principle
experiments in MEKC.

[0128]To confirm the successful generation DNA sequencing fragments, the
cycle sequencing reaction was separated using denaturing polyacrylamide
gel electrophoresis. The results of this separation may be found in FIG.
13. The left most lane represents the migration behavior of a 10 by DNA
ladder. This particular ladder is composed of double-stranded DNA
fragments in increments of 10 by from 20 to 100 by in length. It is clear
that there is not a uniform spacing between the 10-base fragments
generated through the denaturation process prior to introduction to the
gel. Additionally, there appear to be more than 10 bands present in the
lane. This is most likely explained by differing migration of the
individual strands of the double-stranded fragments once they are
denatured. The lowest band in the lane is the 20-base fragment and the
uppermost band corresponds to a molecular weight of 100 bases. This
provides a qualitative indication of the relative lengths of DNA bands
that are electrophoresing in other lanes. The C18-primer may be
found at the far right of the gel, and was loaded at an identical
concentration to the C18 primer in the cycle sequencing reaction.
The center lane represents the results from the cycle sequencing reaction
itself. Although the resolution of bands is poor, there are certainly a
significant number of sequencing fragments present in the mixture. This
particular gel is not expected to successfully resolve sequencing
fragments longer than 20-25 bases. This is due to the small size of the
gel itself. This particular gel is just over 7 cm long and in order to
achieve adequate separation between fragments, this experiment would have
to be repeated on a substantially larger sequencing gel 40 to 50 cm in
length. Nonetheless there is still adequate evidence of the successful
generation of distinct sequencing fragments. Closer inspection of the
dark band immediately below the wells indicates the presence of two
different populations. These two populations represent residual template,
which is ˜7200 bases long, and the full length extension product
expected to be ˜6300 bases. It is difficult to ascertain the
relative amounts of each of the populations present within the gel. One
of the major complications prohibiting quantification, in addition to
inadequate resolution, is the fact that increasingly longer DNA fragments
take up an increasing amount of the stain used for visualization. Also,
the presence of faint bands latitudinal across the entire gel is evidence
of inadequate filtering of infrared light generated by the
transilluminator. The major purpose of this experiment was the
confirmation of the ability to produce a mixture of DNA fragments of
varying lengths from the extension of the aDNA sequencing primer and this
particular gel is evidence of this.

[0130]Prior to the separation of DNA sequencing fragments according to the
invention, it was necessary to determine whether the alkylation of the
DNA primer had a significant impact on the extension ability of the
polymerase typically used in conjunction with the instrument. This was
performed on an ABI Prism 310 Genetic Analyzer, (Applied Biosystems,
Foster City, Calif.). The capillary used was a 50 μm I.D. fused silica
capillary (Polymicro Technologies, Phoenix, Ariz.), 61 cm total length,
50 cm length to detector and maintained at a temperature of 50° C.
The sieving matrix employed was POP6 polymer (Applied Biosystems).
Electrokinetic injection (2.5 kV for 30 seconds) was used to introduce
the DNA sequencing fragment mixture into the capillary. Electrophoretic
separation was conducted under reverse polarity (from cathode to anode)
with an electric field strength of 200 V/cm.

[0131]The ABI Prism 310 Genetic Analyzer is a four-color, capillary gel
electrophoresis-based DNA sequencing instrument. The specific benefit of
a four-color instrument is that the sequencing fragments can be separated
simultaneously. This is done through the use of chain terminators that
are each labeled with a different fluorophore. As a result, the
particular identity of the terminal base of a fragment is specific to a
unique fluorophore and provided the detector signal can be spectrally
filtered properly, the simultaneous detection of each of the four
terminators is possible. Another key feature of the chain terminators
commonly used by the ABI 310, BigDye terminators, is the fact that they
are all excited with the same laser light. The dyes attached to the chain
terminators are not simply a single fluorophore, but rather a pair of
FRET-coupled fluorophores separated by a short flexible liker. Each of
the FRET pairs share a common donor fluorophore but the acceptor
fluorophore it is attached to is unique for each of the four chain
terminators. As a result, each terminator can be excited by laser light
at 488 nm, but they emit light at varying wavelengths, from ˜520 to
˜625 nm. The instrument's detector is essentially a color CCD, and
"virtual" filters within the software filter out any bleed-through
between different channels. In an effort to determine the compatibility
of aDNA sequencing primers with the chain terminators and enzymes used by
the instrument, a cycle sequencing reaction was performed with two
different sequencing primers. The two primers shared an identical length
and sequence, only differing in the attachment of a C18 aliphatic
group, and were each used to sequence the M13mp18 ssDNA template.
Following the cycle sequencing reaction, the samples were purified to
remove residual BigDye terminators and subsequently separated by CGE
using the POP6 polymer designed for DNA sequencing applications.

[0132]Analysis of BigDye-terminated sequencing fragments was conducted by
capillary gel electrophoresis. The capillary used was a 50 μm I.D.
fused silica capillary (Polymicro Technologies, Phoenix, Ariz.), 61 cm
total length, 50 cm length to detector and was maintained at a
temperature of 50° C. 0.1% w/v POP6 polymer (Applied Biosystems)
was used to suppress EOF. Under these conditions, POP6 was seen to reduce
the magnitude of EOF from 4.5×10-4 to 0.08×10-4
cm2/Vs. Electrokinetic injection (2.5 kV for 30 seconds) was used to
introduce the DNA sequencing fragment mixture into the capillary.
Electrophoretic separation was conducted under reverse polarity (from
cathode to anode) with an electric field strength of 200 V/cm. Typical
analysis time was 2 hours.

[0134]Hydrodynamic injection (0.5 psi for 5 seconds) was used to introduce
sample into the capillary. Electrophoretic separation was conducted under
normal polarity (from anode to cathode) with an electric field strength
of 700 V/cm. LIF detection of the C16-Bodipy-F1 labeled sequencing
fragments was performed with excitation/emission wavelengths of 488/520
nm. The capillary coolant temperature was maintained at 22° C. and
samples were stored at 10° C. in DI water prior to injection.

[0135]Following the electrophoretic separation, the sequencing analysis
software spectrally filtered the raw data, and employed a base calling
algorithm to determine the sequence of the DNA templates used in the two
preparations. An abbreviated portion of the sequencing traces, including
the called bases for each trace, can be found in FIG. 15. The lower curve
represents the called bases from base 200 to base 300 for the
C18-cDNA, the upper curve representing the unlabeled fDNA. Not only
were the called bases over this entire region identical, the peak shapes
and peak intensities were virtually identical between the two samples,
indicating no termination bias between the unlabeled and the
C18-labeled sequencing primers. In addition to the 100-base region
depicted in FIG. 15 the two sequences were 99% homologous over a 600 base
stretch, deviating by only 2 called bases.

Example 6

[0136]In the experiment to test separating DNA by transiently attaching a
drag-tag to an allylated DNA analog, a running buffer comprises a
drag-tag having a micelle structure was used. The micelle was formed with
Triton X-100 from Fluka. Stock solutions of Triton X-100 were prepared by
vortexing a suitable amount of Triton in 50 mM Tris MES buffer, pH 8.0,
to arrive at a stock concentration of 48 mM. Aliquots were prepared at
concentrations ranging from 1.2 to 48 mM, vortexed, and centrifuged to
remove bubbles. The Tris MES buffering system was chosen in an effort to
minimize fronting of the DNA peak caused by electrodispersion. Tris HCl
and Tris acetate buffering systems were also investigated but produced
significant peak distortion for high DNA concentrations. Notwithstanding
this distortion, Tris HCl and Tris acetate may be used as alternative
buffering systems, although Tris MES is preferred.

Example 7

[0137]Following successful sequencing of the M13mp18 template with an
alkylated DNA primer using capillary gel electrophoresis, the alkylated
DNA primer was used to synthesize an alkylated DNA analog for use in a
separation modality while transiently attaching drag-tags to the
alkylated DNA analog. Specifically, the DNA analogs were separated by
free-solution electrophoretic in the presence of non-ionic Triton X-100
micelles.

[0138]The molecule, a DNA analog, was synthesized by PCR. The primer used
in the PCR reaction was a C12-Bodipy-Fl-aDNA primer, and the
reaction was carried-out in the presence of acyclic-ATP chain
terminators. Acyclic nucleotide triphosphates are nucleotide analogs that
lack the cyclic sugar group of a standard deoxyribonucleotide. As a
result, they lack the 3' OH functionality required for elongation by
polymerase enzymes. This particular chain terminator was selected due to
its well documented incorporation kinetics with the Therminator enzyme
but, provided the dNTP/chain terminator ratio is chosen appropriately,
the results of most chain terminator chemistries are expected to be
comparable. Following the sequencing reaction, the samples were desalted
and resuspended in distilled water prior to injection in the capillary
electrophoresis instrument. The results of the separation can be seen in
FIG. 12. The two sequences deviated by only 2 called bases for over 600
bases (99% Homologous).

[0139]The lowest electropherogram, FIG. 12(A), represents the separation
of the sequencing fragment in the surfactant-free running buffer. The
tallest peak is expected to correspond to single-stranded DNA, most
likely the primer. The second population at 3.8×10-4
cm-2/Vs corresponds to double-stranded DNA resulting from various
sequencing fragments hybridized to the M13mp18 template.

[0140]The addition of 48 mM Triton X-100 to the running buffer, FIG.
12(B), demonstrates the successful alkylation of sequencing fragments of
various lengths. The broad peak at approximately -3.2×10-4
cm2/Vs represents the double-stranded DNA. More importantly however,
this electropherogram demonstrates the presence of multiple peaks,
presumed to be single-stranded in nature, that show a significant degree
of interaction with the micellular phase. These are the fragments between
-1.5 and -2.5×10-4 cm2/Vs. Thus, it is clear that the
cycle sequencing reaction is producing fragments of different lengths,
and the presence of micelles in the running buffer impacts the
electrophoretic mobility of these fragments.

[0141]Additionally, the magnitude of the peaks for larger fractions is
quite small. This is most likely a result of the fact that this
particular instrument is not specifically designed for the extremely low
fluorescent signals generated from these sequencing fragments. As a
result, a capillary electrophoretic system designed specifically for DNA
sequencing applications was used to investigate the use of micelles for
the separation of sequencing fragments.

[0142]Although data collection followed a protocol identical to that of
the capillary gel electrophoresis sequencing separation, the non-standard
peak spacing rendered it difficult for the current version of the
sequence analysis software to accurately identify the bases. The base
caller employed by the analysis software requires near constant peak
spacing to accurately determine the sequence of basis separated. The
migration time of a DNA fragment of length LDNA is given by

Rather than 1 increasing linearly with LDNA, t scales as 1/LDNA.
As a consequence, the processing of the electropherogram must be done
manually. The first and most important step in the processing of the raw
intensity vs. time data is to spectrally filter the signals as discussed
previously. To do this, the matrix file associated with the dye, in this
case, the BigDye v3.1 matrix, was used. The matrix file represents a
matrix of values that indicate the normalized intensity of each of the
four dyes (the columns of the matrix) in each of the four virtual filters
(the rows of the matrix) and is stored in the instruments software. The
matrix used for this particular data set was:

[0143]Here, the diagonal elements of the matrix are equal to 1. This is
equivalent to stating that the normalized intensity of the blue dye
measured in the blue filter for example, is equal to 1. Also, as one
moves away from the diagonal, the normalized intensities decrease. This
is equivalent to saying that the normalized intensity of blue dye
measured in the green channel (0.23) would be higher than the intensity
of the blue dye measured in the yellow channel (0.01). Once the matrix is
known, each of the four channels of raw data can be normalized for the
contribution of its total intensity from each of the four channels,
eliminating spectral overlap from the data. Mathematically speaking, this
process is equivalent to:

[0144]Following spectral deconvolution, the data is passed through a
moving median filter to eliminate noise in the electropherogram and
finally a unique constant intensity is subtracted from each of the
channels bringing the baseline of each to a value of 0. After the
fluorescence intensity is normalized, the migration time axis can be
converted to allow comparison with measurements discussed previously.
This is accomplished by applying the following equation

μ app = 1 D 1 T Vt . ##EQU00022##

The result of the normalization procedure has been shown in FIG. 16. This
particular set of electropherograms is the result of a micellular-aDNA
interaction during separation for the same set of sequencing primer
investigated sequenced via capillary gel electrophoresis, discussed
above. Only the "C" channel has been displayed for clarity. In FIG.
14(A), the addition of the aliphatic tail is shown to have a profound
impact on the measured electrophoretic mobility.

[0145]Not only are there a substantial number of generated fragments, but
there is significant resolution between a large number of the populations
generated. The electrophoretic mobility of the smallest fragment is
approximately -0.4×10-4 cm2/Vs. This is substantially
lower than the value expected for even the shortest length possible
within the mixture, 25 bases. The experiments have determined that
ss-aDNA interacting with a Triton micelle should carry a hydrodynamic
drag equivalent to an α value of 67.2±0.7 bases. Assuming that
μoaDNA=-2.95×10 cm2/Vs, this predicts that the
lowest possible effective mobility (equivalent to covalent attachment of
a Triton micelle) for a 25 base aDNA would be -0.8×10-4
cm2/Vs. Thus, additional effects are at work other than those
explained by the transient attachment of a Triton micelle as a drag-tag.
In the absence of an alkylation, DNA sequencing fragments are expected to
have a net electrophoretic mobility of 0. In FIG. 16(B), the
electropherogram for the non-alkylated DNA primer, the lowest measurable
effective mobility of the fDNA is approximately -1.15×10-4
cm2/Vs. This could only result from one of two probable scenarios.
First, the fixed polarity of the instrument, cathode to anode, requires
that the separation be conducted under conditions of suppressed EOF. In
order to suppress EOF, a small percentage (0.1% w/v) of POP6 polymer is
added to the surfactant running buffer. This practice is well established
in the literature and it has been observed that the presence of the
polymer at such a low concentration does not induce sieving effects in
free solution. It is possible, however, that the interaction of the
polymer with the Triton micelles induces some strange structure that
leads to increased degrees of hydrodynamic friction. It is also possible
that the fluorophore itself is interacting with the micellular phase
favorably resulting in a decreased electrophoretic mobility.

[0146]Comparison of FIG. 14(B) with the other three channels from the same
separation indicate a strong dependence of the effective electrophoretic
mobilities of the migrating fragments on the fluorophore identity, even
in the absence of the aliphatic group. This strong dependence of
fluorophore identity on the migrational behavior of the fDNA is expected
to be present for the aDNA as well. As a consequence, each of the four
channels was normalized to remove any dependence on fluorophore identity.
Through comparison with the known sequence of the M13mp18 template, the
migration time and hence electrophoretic mobility at the peak maximum of
each fragment that passes by the detector was determined. The resulting
plot of effective mobility vs. expected DNA length can be seen in FIG.
15. The dependence of the fluorophore mobility is clear since each of the
fits has a different slope. While a linear fits seemed to provide the
best fit over this particular range of DNA fragment lengths, a linear
dependence is not expected from the ELFSE equation

μ eff = μ fDNA L DNA L DNA + α .
##EQU00023##

The effective mobility of each fragment should scale hyperbolically with
LDNA rather than the observed linear slope. This is further evidence
that the physical mechanism responsible for the separation is not in fact
consistent with the transient attachment of a Triton X-100 micelle to an
alkylated DNA. The process is assuredly mediated by the presence of the
aliphatic group with the micelle, but separation does not take place
based solely through drag-tag induced increases in the tag's hydrodynamic
radius. However, there still is a sufficient degree of resolution between
the various fragments so a rudimentary sequence analysis should still
provide adequate sequence information. To accomplish this, each of the
electropherograms should be aligned such that the impact of the
fluorophore is taken into account. This can be accomplished by converting
the x-axis in FIG. 15 from electrophoretic mobility to length. The result
of this transformation can be found in FIG. 16. This represents a region
of the set of electropherograms normalized to remove fluorophore impact
on the effective mobilities of the individual fragments. The sequence of
this 30 base region of the M13mp18 template was determined manually. The
bases can be found along the top of the figure and they match the
expected sequence exactly. Although the exact mechanism for the linear
relationship between electrophoretic mobility and fragment length is not
firmly understood, it is most likely induced by either the presence of
the EOF-suppressing polymer or the non-zero partitioning of the BigDye
fluorophores to the surfactant micelles.

[0147]Thus, it was also demonstrated that the use of a large,
transiently-attached drag-tag could enable ELFSE methods to compete with
existing capillary gel electrophoresis-based technologies. The alkylation
of the sequencing primer has no detectable impact on the fidelity of the
sequencing reaction. The normalization of the electrophoretic mobility
into length permitted the successful determination of the DNA fragment's
sequence over a 30-base range.

Hypothetical Example 1

[0148]The lipophilic moiety can be bonded at many positions on a nucleic
acid or nucleoside. For example, one could incorporate amine-containing
nucleosides into the primer, or enzymatically incorporate
amine-containing nucleosides into the cycle-sequencing products or PCR
products. Such schemes could include nucleosides such as amino-allyl
dUTP, aha-dCTP, aminohexyl-dCTP, and amino-butyl-aATP. Additionally, one
could use hydrophobically modified fluorophores attached to the 3' end.
Such fluorophores may be linked to ddNTP chain terminators and may
contain alkyl groups. Those alkyl groups can have a hydrophilic spacer to
minimize the impact of micelle binding on fluorescence signal. Such
spacers may be composed of ethylene glycol subunits.

[0149]One of ordinary skill in the art would recognize other methods of
linking a lipophilic moiety to the nucleic acids.

Hypothetical Example 2

[0150]Proteins can also be separated by molecular weight by transiently
interacting a drag-tag with a protein-detergent complex during a
separation modality. In contrast to the example using nucleic acids, the
lipophilic moiety is not covalently bonded to the protein. Instead, an
ionic bond binds the protein and lipophilic moiety. For example, a sample
protein may be treated with a detergent such as sodium dodecyl sulfate
(SDS). The SDS encases at least a portion of the protein, thereby forming
a protein-detergent complex where the protein is coated with the SDS. The
protein-detergent complex can then be placed in an electrophoresis
device. The electrophoresis device has a running buffer. The running
buffer can comprise 1 mM Triton X-100 in 50 mM Tris-MES at pH 8.0, and
the electric field used can be 300 V/cm. Transient interactions of the
Triton X-100 drag-tags in the running buffer with the SDS-protein
complexes may lead to shifts in electrophoretic mobility that are
molecular weight dependent. A similar experiment can be performed using
Triton X-100-stabilized carbon nanotubes at 0.1 weight % as drag-tags in
a 50 mM Tris-MES buffer at pH 8.0.

Hypothetical Example 3

[0151]In some embodiments, the invention is a method of measuring a
hydrodynamic radius of a drag-tag. For example, Triton X-100-stabilized
carbon nanotubes could be dispersed at 0.1 wt % in a buffer containing 50
mM Tris-HCl at pH 8.0. This buffer could be used as the running buffer as
described above. A sample of alkylated DNA containing a single component
of known molecular weight (number of bases) could be electrophoretically
separated in the above carbon nanotube running buffer. Electrophoretic
separation of the alkylated DNA in the presence of the carbon nanotube
drag-tags should yield one distinct peak whose elution time (or velocity
in a given electric field) reveals the hydrodynamic radius of the carbon
nanotube drag-tags. The above assumes that a large number of transient
interactions between the alkylated DNA and the surfactant-stabilized
carbon nanotubes occur so that polydispersity in the carbon nanotube
drag-tags is not evident.

[0152]Another embodiment of the invention is a method of separating
drag-tags having different hydrodynamic radiuses. This embodiment
comprises using a molecule, such as an alkylated DNA, that binds tightly
to the carbon nanotubes, so that dynamic exchange does not occur. In this
case, the polydispersity of the carbon nanotube population would be
revealed by the presence of multiple peaks in the presence of the single
alkylated DNA population with highly uniform molecular weight.
Fractionation of the peaks could be used to separate carbon nanotubes
based on their hydrodynamic radius. Other colloidal particles could be
characterized and/or separated by a similar scheme, including liposomes,
micelles, proteins, biomolecules, viruses, single-walled carbon
nanotubes, multi-walled carbon nanotubes, oil-in-water emulsions, and
solid particles coated with a surfactant.

[0153]The specific embodiments disclosed herein are not considered to be
limiting. One of ordinary skill in the art would appreciate that
alternative molecules, drag-tags and/or colloid particles could be used,
and that the invention has alternate utilities.

[0154]Such an artisan would recognize that the invention, which generally
relates to transiently binding a drag-tag to a molecule that is being
separated, can function with other molecules, drag-tags, and lipophilic
moieties. It is to be understood that the invention may assume
alternative variations and step sequences, except where expressly
specified to the contrary. It is also to be understood that the specific
embodiments described in the following specification are exemplary of the
invention.

[0155]The present invention is not limited to using nucleic acid analogs
or protein-detergent complexes. One of ordinary skill in the art would
recognize that any molecule that can be separated by various modalities
could be used, so long as the molecule can move at a different rate than
the drag-tag during the separation modality, and can transiently interact
with at least a portion of the molecule with the drag-tag during the
separation modality. Along these lines, although Triton X-100 was
specifically discussed, one skilled in the art would recognize that other
non-ionic surfactants could be used in the formation of the drag-tag.
Additionally, cationic, anionic or zwitterionic could likewise be used,
provided that the drag-tag's mobility was different from the molecule's
mobility.